Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level

doi:

Figure Lengend Snippet: U87 and T98G cells were treated with 200 μM TMZ. At 72 h, real time PCR was performed for PTCH1 mRNA (A) and western blot for PTCH1 and SHH (precursor (P) and N-terminus (N)) and β-actin. The values for the untreated group were assigned 1 and are represented by an open bar. The changes in the treated cells were presented as fold change over the vehicle-treated cells. (B) U87 and T98G were knocked down for SHH with siRNA and then analyzed for SHH (precursor, P and mature, N) and β-actin by western blot. (C) The knocked down cells in ‘C’ were treated with TMZ. At 72 h, the cells were assessed for viability with Cell Titer Blue. The data are presented as the mean % viability relative to vehicle treated siRNA transfected cells ±SD, n = 4. (D) U87 and T98G were knocked down for Dicer1 . Control was transfected with non-targeting oligo. Western blot was performed for Dicer. The membrane was stripped and reprobed for β-actin. (E) The cells in ‘E’ were treated with 200 μM TMZ. At 72 h, western blot was performed for PTCH1. The membrane was stripped and reprobed for β-actin (F) and assessed for viability. (G) p < 0.05 vs. control and non-targeting oligo.

Article Snippet: Rabbit anti-human PTCH1 was custom-ordered from Novoprotein (Short Hills, NJ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level

doi:

Figure Lengend Snippet: (A) Computational analyses were performed for potential miRNA interacting site with the PTCH1 3′ UTR. The alignment between the sequence for miR-9 and the 3′ UTR of PTCH1 is shown below. (B) U87 and T98G cells were treated with 200 μM TMZ. At various times, real time PCR (Taqman) was performed for miR-9. (C) The studies in ‘C’ were repeated, except for studies at the 72-h time point, and with primers for miR-9-2, using Sybr-Green. (D) U87 and T98G were transfected with non-targeting oligo or anti-miR-9 and then treated with 200 μM TMZ. After 72 h, cell viability was assessed and presented as % viability, n = 4 ±SD. (E) The heat map shows the analyses from 505 miRNA arrays with GBM tissues from The Cancer Genome Atlas (TCGA). The map represents the miRNAs with +/– 0.5-fold changes from the internal control. Arrow highlights the output for miR-9. * p < 0.05 vs. other time points, ** p < 0.05 vs. vehicle. *** p < 0.05 vs. non-targeting oligo.

Article Snippet: Rabbit anti-human PTCH1 was custom-ordered from Novoprotein (Short Hills, NJ).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transfection

Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level

doi:

Figure Lengend Snippet: (A & B) CCL64, stably transfected with pPTCH-UTR-JM, was transiently transfected with pre-miR-9, non-targeting oligo and/or target protection (TP) oligo. After 48 h, luciferase activities were quantitated and then presented as normalized luciferase ±SD, n = 4; TP: Target Protector (A). Western blots were performed with whole cell extracts for PTCH1 and β-actin. The normalized band densities are shown at the bottom of the images (B). (C) U87 and T98G, stably transfected with pPTCH-UTR-JM or pPTCH-UTR™-JM, were transiently transfected with anti-miR-9 and then treated with 200 μM TMZ or vehicle. After 48 h, luciferase activities were quantitated and the results are presented as mean RLU ±SD, n = 4. (D & E) MiR-9 was ectopically expressed in U87 and T98G cells. Total RNA was isolated and then studied by real time PCR for PTCH1 mRNA (D) or Gli1 (E). (F) Diagram show increased miR-9 in TMZ-treated GBM cells leading to activated SHH signaling. (G) Western blots with whole cell extracts were performed for Gli1, PTCH1 and β-actin using miR-9-transfected or vector-transfected GBM cells. (H–K) U87 and T98G cells were ectopically expressed for miR-9 or transfected with vector alone. Real-time PCR was performed for MDR1 (H) and ABCG2 (I). The values for control vector in the real time were normalized to 1 for fold-change of the miR-9 transfectants, mean±SD, n = 4; flow cytometry for membrane MDR1 (J) and ABCG2 (K). * p < 0.05 vs. Pre-mir-9 and TP, ** p < 0.05 vs. pPTCH-UTR-JM. *** p < 0.05 vs. vector alone.

Article Snippet: Rabbit anti-human PTCH1 was custom-ordered from Novoprotein (Short Hills, NJ).

Techniques: Stable Transfection, Transfection, Luciferase, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Plasmid Preparation, Flow Cytometry

Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level

doi:

Figure Lengend Snippet: (A) Real-time PCR was performed for the miRNA, predicted for PTCH1 (Figure ). The results are presented as the mean fold change ±SD, n = 4. (B) Real time PCR for miR-9-2 was performed in four independent studies using RNA from BT145 and BT164 cells. The results of BT145 were normalized to 1 and then used to calculate the fold change for the values of BT164, mean±SD. (C) Real time PCR were performed for PTCH1 as for ‘B’ and the results are similarly presented. (D) Whole cell extracts from BT145 and BT164 were studied by western blot for PTCH1, SHH-P (precursor), SHH-N (mature) and β-actin. (E) Real time PCR was performed for MDR1 and ABCG2 mRNA with total RNA from BT145 and BT164. (F & G) Flow cytometry was performed for membrane P-glycoprotein ( MDR1 ) (F) and ABCG2 (G). The right panels show the overlay between the two patients. * p < 0.05 vs. BT145.

Article Snippet: Rabbit anti-human PTCH1 was custom-ordered from Novoprotein (Short Hills, NJ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry

Journal: Cells

Article Title: Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency

doi: 10.3390/cells7080107

Figure Lengend Snippet: Modes of signaling in Hedgehog (Hh) pathway-dependent cancer.

Article Snippet: As the Hh receptor Ptch1 is an Hh target gene, this receptor has been shown to be overexpressed in many cancers such as lung, breast, prostate, ovary, colon, brain, melanoma [ , , ], and myeloid leukemia [ , ] (see the Human Protein Atlas website http://www.proteinatlas.org/ENSG00000185920-PTCH1/cancer , [ ]) ( ).

Techniques: Activation Assay

Journal: Cells

Article Title: Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency

doi: 10.3390/cells7080107

Figure Lengend Snippet: Ptch1 protein level in cancers. From the Protein Atlas website http://www.proteinatlas.org/ENSG00000185920-PTCH1/cancer .

Article Snippet: As the Hh receptor Ptch1 is an Hh target gene, this receptor has been shown to be overexpressed in many cancers such as lung, breast, prostate, ovary, colon, brain, melanoma [ , , ], and myeloid leukemia [ , ] (see the Human Protein Atlas website http://www.proteinatlas.org/ENSG00000185920-PTCH1/cancer , [ ]) ( ).

Techniques:

Journal: Cells

Article Title: Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency

doi: 10.3390/cells7080107

Figure Lengend Snippet: Ptch1 sequence homologies with the Niemann-Pick disease type C1 protein (NPC1) and the bacterial transporters from the RND family such as AcrB, MexB and CzcA. ( A ) Protein topology. The sterol sensing domains (SSD) are represented in red, and the transmembrane segment containing the highly conserved GXXXD motif is indicated. ( B ) Sequence alignment of the highly conserved GXXXD motif.

Article Snippet: As the Hh receptor Ptch1 is an Hh target gene, this receptor has been shown to be overexpressed in many cancers such as lung, breast, prostate, ovary, colon, brain, melanoma [ , , ], and myeloid leukemia [ , ] (see the Human Protein Atlas website http://www.proteinatlas.org/ENSG00000185920-PTCH1/cancer , [ ]) ( ).

Techniques: Sequencing

Journal: Cells

Article Title: Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency

doi: 10.3390/cells7080107

Figure Lengend Snippet: Ptch1 cholesterol transport activity regulates Hedgehog signaling. Ptch1 is represented in green, Smoothened in red, cholesterol in yellow, and Shh as a golden ball.

Article Snippet: As the Hh receptor Ptch1 is an Hh target gene, this receptor has been shown to be overexpressed in many cancers such as lung, breast, prostate, ovary, colon, brain, melanoma [ , , ], and myeloid leukemia [ , ] (see the Human Protein Atlas website http://www.proteinatlas.org/ENSG00000185920-PTCH1/cancer , [ ]) ( ).

Techniques: Activity Assay

Journal: Cells

Article Title: Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency

doi: 10.3390/cells7080107

Figure Lengend Snippet: Combination of Ptch1 drug efflux inhibitor and chemotherapy is a potential new therapeutic option to overcome the drug resistance of cancer cells. Ptch1 inhibitor (in green) inhibits the doxorubicin (in red) efflux activity of Ptch1 (in blue). This allows the concentrations of doxorubicin to be reached that are required to kill cancer cells and fight chemotherapy resistance.

Article Snippet: As the Hh receptor Ptch1 is an Hh target gene, this receptor has been shown to be overexpressed in many cancers such as lung, breast, prostate, ovary, colon, brain, melanoma [ , , ], and myeloid leukemia [ , ] (see the Human Protein Atlas website http://www.proteinatlas.org/ENSG00000185920-PTCH1/cancer , [ ]) ( ).

Techniques: Activity Assay